ChemBioChem

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

Flavins are organic redox cofactors central to metabolism and uniquely capable of acting as extracellular electron shuttles. For life to have emerged, it must have disengaged itself from its stationary geochemical environment, a step requiring mobile redox-active components. The role of flavins at life's origin has been debated for decades, centered on their capacity for both one- and two-electron chemistry, distinguishing them from nicotinamides and iron-sulfur clusters. Here we chart the abiotic reduction of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin under hydrothermal conditions (40 {degrees}C, 1 bar N2 or 5 bar H2, pH 6, 8, and 10) by nickel (Ni) and iron (Fe). Flavins show greater environmental versatility than hydride carriers such as NAD and can harvest electrons from metals that would otherwise reduce water's protons to H2. Reduction is favoured under acidic conditions, while increasing molecular charge at higher pH impedes electron transfer. Ni acts as a hydrogenation catalyst, reducing deprotonated flavins via hydride transfer, suggesting mineral composition could have influenced geochemical selection of early electron carriers. Reduced FMNH2 and FADH2 were tested as electron shuttles toward Fe3+-containing minerals, revealing that FMNH2 enables faster mineral dissolution than FADH2. We further demonstrate complete redox cycling of FMN through Ni-assisted H2 reduction and subsequent oxidation by magnetite (Fe3O4) under inert atmosphere, releasing Fe2+. This study highlights the versatility, stability and redox chemical capabilities of flavins in prebiotic context.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biocatalytic metal-containing clusters are nanometer-sized chemical reactors that enable enzymes to perform chemistry far beyond what would be possible with amino acids alone 1,2. These clusters display a remarkable variability3 and elucidating their exact mechanisms is often difficult to determine, requiring input from a multitude of techniques ranging from spectroscopic to structural and theoretical methods. Thus, for unknown clusters, it is essential to accumulate, collate, and interpret as much information as possible from every relevant technique available. Here we report the discovery and in-depth, interdisciplinary characterization of an entirely novel, [Cu-4Fe-4S] cluster in the protein acetol dehydrogenase (AceDH). AceDH, isolated directly from Methylacidiphilum fumariolicum SolV cells, catalyzed the oxidation of acetol to methylglyoxal, proving its role in the 2-propanol/acetone metabolism of methanotrophs4-6. Structural, spectroscopic and electrochemical analyses reveal the [Cu-4Fe-4S] cluster has a unique three-dimensional- and electronic structure involving electronic coupling between the copper and one of the iron atoms, likely contributing to its high, +275 mV, redox potential. The binding site for the novel cluster is composed of two protein subunits and involves a novel motif. These findings expand the known repertoire of biological metal cofactors and provide insight into how heterometallic clusters are adapted for biological processes.

L-DOPA is a key therapeutic agent for Parkinsons disease, with growing demand due to global population aging. Here we report that heme-dependent tyrosine hydroxylase (TyrH) can utilize an ascorbate/O2 system--as an alternative to H2O2--to synthesize L-DOPA with markedly enhanced operational stability. While exogenous H2O2 rapidly inactivates TyrH within minutes, sodium ascorbate (NaAsc) enables sustained catalysis for up to 24 h, surpassing the H2O2-driven yield after only 30 min. UV-vis spectroscopy confirms that H2O2 readily degrades the heme center, whereas the heme remains intact in the presence of NaAsc. QM/MM simulations reveal that in situ generated H2O2 leads to the active species of Compound I for tyrosine hydroxylation. Through systematic optimization, we establish efficient reaction conditions (40 {micro}M TyrH, 1 mM L-Tyr, 100 mM NaAsc, pH 8.5, 40 {degrees}C), achieving >95% conversion of L-Tyr to L-DOPA within 2 h. This work not only provides a robust and sustainable biocatalytic route for L-DOPA production but also highlights the broader applicability of the ascorbate/O2 pathway in heme-enzyme catalysis.

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

Modern protein design methods based on deep learning allow generation of customized protein scaffolds with diverse geometries and functionalities. Here, we capitalize on these recent advances to develop hyper-thermostable de novo CO2 reductases featuring a cobalt porphyrin IX cofactor (CoPPIX). CoPPIX containing enzymes were assembled in vivo through media supplementation with cobalt salts and assessed for photocatalytic CO2 reductase activity. We identified two cysteine-ligated designs that exhibit high activity (>1000 turnovers at rates of up to 25 min-1) while suppressing competing hydrogen evolution pathways. A 2.1 [A] crystal structure shows close agreement to the design model with the Co-Cys bond programmed as intended. This study showcases the power of computational protein design in developing artificial enzymes to activate challenging molecules such as CO2.

Designing redox proteins with predictable and tuneable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non-natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tuneable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

Norepinephrine (NE) is a key neurotransmitter and hormone involved in diverse physiological and pathological processes. Beyond its canonical non-covalent signaling through adrenergic receptors, NE also induces protein post-translational modifications (PTMs), representing an emerging regulatory mechanism. Two major forms of NE-derived PTMs have been identified: non-enzymatic norepinephrinylation (NEylation) of cysteine residues mediated by NE quinone and transglutaminase 2 (TG2)-catalyzed NEylation of glutamine residues. However, the biochemical basis and pathophysiological roles of NEylation remain poorly understood due to limited detection tools. Here, we report a bioorthogonal reaction for selective labeling and enrichment of the NEylation proteome in cell lines and tissues, which is based on acid-catalyzed dehydration and 1,6-addition to thiol probes. This strategy enables fluorescence imaging and chemical proteomic profiling, revealing NEylation as a widespread PTM that affects enzymatic activities of modified proteins, including protein tyrosine-protein phosphatase non-receptor type 11 (PTPN11). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/731782v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19dce3aorg.highwire.dtl.DTLVardef@14e7f30org.highwire.dtl.DTLVardef@80354aorg.highwire.dtl.DTLVardef@12ac6f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

We report here a method for the chemical synthesis of Fmocprotected -hydroxyglyine (-OH-Gly) dipeptides. Our method features operational simplicity and is compatible with protecting groups for peptide synthesis. Utility is then demonstrated through substitution at the -OH-Gly position to yield myriad non-natural amino acid-containing dipeptide fragments.

DNA assembly is a cornerstone of synthetic biology, enabling the construction of bespoke genetic systems for applications ranging from metabolic engineering to DNA nanotechnology. Conventional Gibson Assembly (GA), the most widely used method, relies on 5' exonucleolytic resection and elevated temperatures ([~]50 {degrees}C), which together prevent the retention of 5' modifications and restrict compatibility with temperature-sensitive functionalities. Here, we report a DNA assembly strategy, 3 exonuclease-mediated low-temperature DNA assembly (3LTDA), which generates complementary 5' overhangs while preserving 5' end integrity. This approach enables the efficient assembly of blunt-ended, 5'-functionalised DNA fragments into both linear and circular constructs at ambient temperature (21 {degrees}C), with some assembly observed at temperatures as low as 4{degrees}C. We systematically optimise reaction conditions and demonstrate that this method supports efficient plasmid re-circularisation and multi-fragment assembly, including the construction of a [~]12.5 kbp plasmid from multiple DNA components. Comparative analysis across several DNA substrates shows that, under their respective optimal conditions, this approach matches or exceeds GA performance, improving assembly efficiency by up to 12.8%. Sequence analysis confirms high fidelity with no detectable base-pairing errors across assembled junctions. Crucially, this method preserves chemically functionalised 5' termini, enabling downstream conjugation and biochemical functionality. Retention of azide and biotin modifications was verified through fluorescence imaging, bead-based co-localisation, and enzymatic activity in ELISA-based assays. This is in contrast to GA-assembled controls, which showed complete loss of functionality under comparable conditions. We further assembled 5 kbp dsDNA using 3LTDA from four independent segments, three with different fluorescence reporters, and the fourth containing a biotin group for microparticle conjugation, each on the 5 end. Under fluorescence illumination, bead-bound DNA with all three fluorescence markers were detected. Conventional GA assembled constructs, on the other hand, failed to retain the reporter groups and the fluorescent images did not show the presence of any fluorescent markers. In addition to enhanced performance, the method could also reduce reagent cost and eliminate the need for elevated temperatures, simplifying workflows and expanding the applicability of multi-functionalised DNA constructs. Collectively, this work establishes 3LTDA as a robust, low-temperature alternative to conventional GA, with advantages for applications requiring precise chemical modification, temperature-sensitive components, or deployment outside conventional laboratory environments.

Natural product (NPs) and their derivatives are a major source of small-molecule drugs, and the building blocks of these NPs are often amino acids. These include both proteinogenic and nonproteinogenic amino acids (NPAAs), the latter of which expand the structural diversity of NPs. Homologation, or the addition of a methylene group to the amino acid side chain, is one modification that generates NPAAs. If the natural homologation pathway can be characterized and engineered, it could be used to diversify NPs. In this study, we investigated the terminal enzyme of this pathway, HphB, to determine its substrate scope. HphB was tested with various substrates that differed in backbone and/or side chain structures relative to its natural substrate. The results showed that HphB exhibits high promiscuity toward substrates with different side chains while maintaining strict specificity for the substrate backbone. Comparative analysis with two homologous enzymes from primary metabolic pathways revealed that HphB displays markedly higher substrate promiscuity. Bioinformatics analysis and structural modeling suggest that this promiscuity arises from the absence of a "lid" over the active site, resulting in increased solvent exposure of the substrate side chain. This study highlights the unique substrate flexibility of HphB and is a step toward engineering the homologation pathway to generate amino acid derivatives.

Actin turnover is a fundamental cellular process essential for cell dynamics, whose control is critical for both medicine and biotechnology. However, conventional small molecules modifying actin turnover scramble the structure of actin filaments and display high cellular toxicity. MICAL enzymes oxidizing methionine (Met) residues in actin can potentially address this challenge, but their large size and multiple required cofactors make MICALs manufacturing and utilization difficult. Here we show that redox-active chiral decavanadate nanoclusters with tartaric acid are capable of site-selective actin modulation, mimicking MICALs, while requiring no cofactors, displaying high biocompatibility and being membrane permeable. Decavanadate nanoclusters serve as atomically precise "nano-enzymes" oxidizing three Met residues in globular actin, including Met-176; the latter inhibits the opening of the backdoor segment and prevents depolymerization of actin filaments. The structure of actin filaments formed after nanocluster treatment revealed no structural disturbances as confirmed by cryo-electron microscopy. The biocompatibility and bioactivity of chiral decavanadate nanoclusters was demonstrated by modulation of actin in living NG108-15 cells. Taking advantage of atomically precise structure of the nanoclusters, we show that their docking into actin can be predicted computationally, indicating the possibility of programmable actin modulation using the tools of nanochemistry.

Inorganic polyphosphate (polyP) is a ubiquitous phosphate biopolymer involved in diverse cellular processes. Despite its significance, selective detection of polyP remains challenging because of its simple and highly charged structure. Here, we report a near-infrared (NIR) fluorogenic turn-on chemosensor for selective polyP detection and imaging, SiX-DPA-Zn. The probe combines a silicon-xanthene (SiX) fluorophore with a zinc(II)-coordinated 2,2'-dipicolylamine (DPA-Zn2+) recognition unit and shows more than 100-fold selectivity for inorganic polyP over ADP and ATP. SiX-DPA-Zn enables quantitative detection of polyP at micromolar concentrations in microplate assays and stains a broad range of polyP species, starting from tripolyphosphate, in polyacrylamide gels. In HEK293 cells expressing Escherichia coli polyphosphate kinase 1, the probe visualizes intracellular polyP and enables quantitative analysis of polyP levels in relation to nuclear proteins for example fibrillarin and nucleolin. Stimulated emission depletion (STED) microscopy further revealed subdiffraction-sized polyP granules within polyP aggregates. SiX-DPA-Zn is the first near-infrared (NIR) fluorogenic chemosensor for polyP that is compatible with multiple detection platforms, including microplate assays, polyacrylamide gel staining, confocal and super-resolution STED microscopy.

Bacterial microcompartments (BMCs) are a diverse and widespread class of protein-based organelle consisting of a semi-permeable protein shell encapsulating an enzymatic core. Along with their native assembly pathway, isolated BMC shell proteins have been shown to assemble into alternative superstructures such as flat sheets and nanotubes. The self-assembly and modularity of BMC shell proteins make them of great interest as modular platforms for applications involving scaffolding, immobilization and compartmentalization. While the assembly of BMC shell proteins into higher-order structures has been well-studied, the design of controllable and modular cargo loading is underdeveloped in comparison. Recently, we reported the pH-controlled assembly of CcmK2 - the major hexameric shell protein of the {beta}-carboxysome BMC - into monodisperse mesh-like microscale particles. Here, we develop a suite of encapsulation strategies for stochastic or targeted loading of various cargos, as well as the direct conjugation of cargo to CcmK2 particles. Our systematic analysis demonstrates that cargo loading and particle assembly can be modulated by the choice of recruitment strategy and the order of cargo introduction. Our findings also reveal a cooperative cargo loading mechanism during assembly that influences particle sizing and apparent morphology. Our study serves as a blueprint for the rational design of tunable cargo loading into engineered BMC-derived microcompartment systems for diverse biotechnological applications.

The catalytic rate constant (kcat) for product formation is considered a turnover number. Therefore, it is often mistakenly believed that kcat equals the turnover number and the number of substrate molecules changed per unit of time. Therefore, the aim of this study is to show that the rate constant for product synthesis and release is not always the same as the rate constant [Formula] for substrate utilization. To determine the precise substrate concentration at which these two rate constants are identical, it is appropriate to derive equations that allow the computation of [Formula]. In the end, the study will provide the most likely concentration of enzymes that can guarantee minimal or no recycling. An analysis of the literature on invertase (EC 3.2.1.26) and the Bernfeld method of generating Michaelian kinetic parameters for human salivary alpha-amylase (HSAA, EC 3.2.1.1) revealed that all kinetic parameters except [Formula] increased with substrate concentration. Meanwhile, the values for invertase decreased from 0.0697 to 0.0361/min, and the values for HSAA decreased from 5,802.4687 to 3,213.0124/min. The magnitude of [Formula] for each substrate concentration ([ST]) is not always equal, except when [ST] is determined post-assay by computation or extrapolation. The lower [ST] at which [Formula] and kcat for [HSAA] are equal is 3.667540128 g/L (5.682584642 M), which is similar to the molarity of HSAA (5.6101967709 M). The kcat for HSAA was 11,930.9885/min. Future assays should aim to generate large amounts of data for a robust statistical analysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/728646v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@c49b65org.highwire.dtl.DTLVardef@1b60655org.highwire.dtl.DTLVardef@159ba67org.highwire.dtl.DTLVardef@1dce0af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Developing enzymatic mechanisms for C-F bond formation remains a long-standing challenge. Here, we repurposed the biosynthetic nonheme Fe enzyme EgtB, which features a three-histidine facial triad, to catalyze C(sp3)-H fluorination reactions. Directed evolution of EgtB afforded two new-to-nature fluorine atom transferases with opposite enantiopreference, EgtBCHF1 and EgtBCHF2, with up to 28-fold improved total activity. In contrast to our previously evolved nonheme Fe fluorine atom transfer biocatalyst ACCOCHF, which contains a two-histidine-one-carboxylate facial triad, the evolved EgtBCHF variants displayed unexpected hydroxylation activity. 18O-labeling experiments showed that the hydroxy group originated from water rather than residual O2. Computational studies suggested that the three-histidine-supported Fe(III) center exhibits enhanced Lewis acidity compared to the two-histidine-one-carboxylate system, allowing deprotonation of Fe(III)-bound water to form a Fe(III)-OH species to catalyze radical hydroxylation. Primary coordination-sphere mutagenesis in EgtB and ACCO further supported the critical role of Fe coordination chemistry in controlling radical rebound reactivity and selectivity. Computational studies revealed that Fe coordination chemistry strongly influences both fluorine atom abstraction and radical rebound, with the intrinsic C-X (X = F, OH, and N3) bond forming radical rebound preference following the order N3 > OH > F. Furthermore, multivariate linear regression analysis revealed that fluorine atom abstraction is primarily governed by the intrinsic Fe-F bond strength, whereas fluorine rebound is predominantly controlled by the electronic structure of the Fe(III) intermediate. Together, these findings provide mechanistic insights into nonheme Fe enzymology and reprogramming toward selective radical rebound reactions, including challenging C-H fluorination. Table of Contents (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/737789v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1ad85b2org.highwire.dtl.DTLVardef@1248bd4org.highwire.dtl.DTLVardef@58268dorg.highwire.dtl.DTLVardef@14b2da0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sickle cell disease (SCD) is caused by a single amino acid substitution in the {beta}S globin chain at 6th position (6E[->]V). This results in polymerization of deoxy state of sickle hemoglobin (HbS), followed by its precipitation and subsequent sickling of erythrocytes. These deformed cells can block small capillaries (vaso-occlusion), causing cardiovascular complications, ultimately leading to ischemia-reperfusion injury, severe oxygen deficiency, and progressive systemic damage. Occasionally, patients with SCD have been observed to produce exorbitantly high levels of fetal hemoglobin (HbF), which has been linked with the inhibition of HbS polymerization. One of the effects of hydroxyurea, the most commonly used therapeutic for SCD, is to elevate HbF levels. However, the mechanism of inhibitory role of HbF on HbS polymerization is largely unknown. This study attempts to gain insights into the mechanisms involved in this process by means of native mass spectrometry, ion mobility mass spectrometry, and hydrogen deuterium exchange-based mass spectrometry (H/DX-MS). The conformational flexibility of asymmetric hemoglobin, HbFS (2{gamma}{beta}S), for the observed regions in the tetrameric molecule appears to be more in the deoxy state as compared to the oxy state, eventually leading to reduced polymerization of sickle hemoglobin in patients with SCD that express elevated HbF levels.